Immune Cell Infiltration Across Breast Cancer Subtypes: A Marker Gene Analysis of the METABRIC Cohort

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Name: Chung Yan	Surname: Yu
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Declaration of the use of generative AI

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Abstract

The ability for immune cells to infiltrate the tumour microenvironment (TME) plays a key role in the biological war machine taking on breast cancer.

Introduction

Tumour cells evading the detection and wrath of the immune system is a recognised hallmark of cancer¹, and breast cancer is no exception². The interplay between tumour cells, immune cells and other local factors form the tumour microenvironment (TME), and research in the TME of breast cancers has yielded clinical applications³. The amount of tumour infiltrating immune cells in breast cancers has shown prognostic value⁴, and this amount and prognostic impact varies across breast cancer’s molecular subtypes⁵. These subtypes are defined by gene expression⁶, and are popularised and commercialised as the PAM50 gene expression panel, which has its own prognostic significance⁷. The subtypes include luminal A, luminal B, HER2-enriched, basal-like and normal-like.

The METABRIC cohort from Curtis et al.⁸ is one of the larger datasets on breast cancer containing clinical, gene expression and mutation data of 2,509 patients. METABRIC’s analysis has already shown immune infiltration in one of their integrative clusters (IntClust4), showing deletions in T cell receptors and mRNA amplification for immune cell activity, but stopped short of fully characterising the immune cells involved. Jiang et al.⁹ characterised immune cell infiltration in the METABRIC cohort using CIBERSORT¹⁰, developing new immunotypes with prognostic significance. However, CIBERSORT is more of a black box model, a computational approach using deconvolution to estimate cell proportions from RNA mixtures. Therefore there exists a need to use a transparent approach to score immune cell infiltration in METABRIC’s breast tumour samples, and scrutinise any prognostic value from such scoring.

A panel of immune cell marker genes developed by Danaher et al.¹¹ were used to score immune infiltration in the METABRIC cohort. The infiltration of different immune cell types as well as their prognostic value were investigated across the PAM50 subtypes. The goal is to characterise the immune landscape across PAM50 subtypes, identify which immune populations associate with survival, and test whether immune cell scores or multi-dimensional immune profiles improve prognostic value beyond subtype classification alone.

Methods

# To pull fresh data from cBioPortal instead of using the included processed files,
# delete the data/processed/ folder and re-render this document.
source("scripts/00-data-cleaning.R")
source("scripts/01-immune-cell-scoring.R")
source("scripts/02-task1-exploration.R")

This study makes use of the METABRIC dataset⁸ available on cBioPortal^12–14, including clinical, gene expression and mutation datasets of 2,509 patients. For gene expression, the log2 intensity was used in place of the normalised z-score to preserve the proportions when applying the immune cell scoring.

The principle of avoiding circularity guided two data selection decisions. First, PAM50 subtypes were chosen over IntClust as IntClust subtypes are defined by copy number aberrations that can directly influence immune marker gene expression, and IntClust4 is already characterised by immune infiltration. The clear lack of overlap between the PAM50 and Danaher’s immune cell gene panels also strengthened this choice. Second, the exclusion of Claudin-low patients. Claudin-low is another breast cancer subtype used alongside PAM50 subtypes on cBioPortal’s METABRIC dataset, and is characterised by immune infiltration¹⁵. Fougner et al.¹⁵ further demonstrated that Claudin-low is not an independent subtype in addition to the PAM50 subtypes, therefore patients of this category (n = 218) were removed.

Immune cell scoring is done by taking the mean log2 gene expression of the cell types’ marker genes, originally defined by Danaher et al.¹¹ and tabulated in Table S1. Of the 60 marker genes from the Danaher panel, only 58 genes have expression data in the METABRIC dataset. TPSB2 is missing for mast cells and XCL2 for NK cells. The CD4 T cell score is derived by subtracting the CD8 T cell score from the overall T cell score. The Danaher scoring method was chosen for its simplicity and marker genes validated against flow cytometry, in contrast to deconvolution approaches previously applied to this cohort⁹.

Immune variation across PAM50 subtypes was assessed by Kruskal-Wallis test with Wilcoxon for pairwise comparisons. Prognostic associations were evaluated using Cox proportional hazards models, first univariately and then multivariately to adjust for PAM50 subtypes, along with Kaplan-Meier curves with log-rank tests for visualisation. To develop multi-dimensional immune profiles, clusters were fitted via k-means with silhouette scoring. All p-values from multiple testing were corrected via Benjamini-Hochberg false discovery rate (FDR) and reported as FDR throughout. Overall survival was used for prognosis as the disease-specific survival data used by Curtis et al.⁸ was not available on cBioPortal.

Cell type	Markers	Found	Genes used	Genes missing
B-cells	9	9	BLK, CD19, FCRL2, MS4A1, FAM30A, TNFRSF17, TCL1A, SPIB, PNOC	—
CD45	1	1	PTPRC	—
CD8 T cells	2	2	CD8A, CD8B	—
Cytotoxic cells	10	10	PRF1, GZMA, GZMB, NKG7, GZMH, KLRK1, KLRB1, KLRD1, CTSW, GNLY	—
DC	3	3	CCL13, CD209, HSD11B1	—
Exhausted CD8	4	4	LAG3, CD244, EOMES, PTGER4	—
Macrophages	4	4	CD68, CD84, CD163, MS4A4A	—
Mast cells	5	4	TPSAB1, CPA3, MS4A2, HDC	TPSB2
NK CD56dim	4	4	KIR2DL3, KIR3DL1, KIR3DL2, IL21R	—
NK cells	3	2	XCL1, NCR1	XCL2
Neutrophils	7	7	FPR1, SIGLEC5, CSF3R, FCAR, FCGR3B, CEACAM3, S100A12	—
T-cells	6	6	CD6, CD3D, CD3E, SH2D1A, TRAT1, CD3G	—
Th1 cells	1	1	TBX21	—
Treg	1	1	FOXP3	—

NoteTable S1 — Immune marker gene coverage

Verifying the immune marker gene coverage on the Illumina HT-12 v3 microarray of METABRIC dataset.

Results

Cohort Summary and Mutation Landscape

Characteristic	Overall N = 1756 (100%)	Basal-like N = 209 (12%)	HER2-Enriched N = 224 (13%)	Luminal A N = 700 (40%)	Luminal B N = 475 (27%)	Normal-like N = 148 (8.4%)	p-value
Age at diagnosis	62 (52, 71)	54 (44, 65)	59 (50, 69)	63 (53, 72)	67 (59, 74)	57 (46, 66)	<0.001
ER status							<0.001
Negative	339 (20%)	172 (84%)	115 (53%)	13 (1.9%)	11 (2.4%)	28 (19%)
Positive	1,386 (80%)	33 (16%)	103 (47%)	677 (98%)	456 (98%)	117 (81%)
Unknown	31	4	6	10	8	3
Tumour Grade							<0.001
1	154 (9.2%)	2 (1.0%)	4 (1.9%)	118 (18%)	18 (3.9%)	12 (8.6%)
2	714 (42%)	17 (8.3%)	54 (25%)	378 (57%)	186 (41%)	79 (57%)
3	815 (48%)	187 (91%)	155 (73%)	172 (26%)	253 (55%)	48 (35%)
Unknown	73	3	11	32	18	9
Cellularity							<0.001
High	898 (52%)	124 (60%)	125 (59%)	326 (47%)	292 (63%)	31 (23%)
Low	163 (9.5%)	28 (14%)	15 (7.0%)	53 (7.7%)	22 (4.7%)	45 (34%)
Moderate	650 (38%)	54 (26%)	73 (34%)	312 (45%)	153 (33%)	58 (43%)
Unknown	45	3	11	9	8	14
Follow-up (months)	116 (60, 184)	86 (33, 183)	97 (39, 172)	130 (82, 197)	104 (55, 164)	121 (64, 176)	<0.001
Deceased	1,044 (59%)	115 (55%)	157 (70%)	378 (54%)	315 (66%)	79 (53%)	<0.001

NoteTable 1 — Cohort characteristics by PAM50 subtype

Baseline characteristics of 1,756 METABRIC patients with expression data, stratified by PAM50 subtype. 218 claudin-low and 6 NC patients excluded. Continuous variables summarised as median (Q1, Q3); categorical as n (%). P-values from Kruskal-Wallis (continuous) or Pearson’s Chi-squared (categorical) tests.

NoteFigure 1 — Data availability across modalities

Intersection plot showing patient counts for each combination of available data modalities (N = 2,509).

• 529 patients without expression data
• significantly younger than the 1980 with expression data; Wilcoxon rank-sum p < 0.001
• median age 58 vs 61.8 years

The 529 patients without expression data were significantly younger than the 1980 with expression data (median age 58 vs 61.8 years; Wilcoxon rank-sum p < 0.001).

NoteFigure 2 — Mutation-immune cell score associations

Median immune cell score differences (mutated minus wild-type) for genes with at least one significant association across 14 cell types. Significance by Wilcoxon rank-sum test (FDR < 0.05); only genes mutated in ≥10 patients tested.

NoteFigure 3 — TP53 mutation and immune infiltration after PAM50 adjustment

PAM50-adjusted effect sizes from linear regression (x-axis) and significance (y-axis) for each immune cell type. Dashed line marks FDR = 0.05.

Immune Infiltration Across PAM50 Subtypes

NoteFigure 4 — Immune cell score heatmap across PAM50 subtypes

Z-scored immune cell scores for 1,756 patients, split and hierarchically clustered by PAM50 subtype. Blue = below-mean; red = above-mean infiltration.

NoteFigure 5 — Immune cell scores by PAM50 subtype

Violin plots with box plots for cell types reaching Kruskal-Wallis FDR < 0.05. Lettering above each subtype plot indicate similarity/difference, subtypes sharing a letter do not differ significantly (pairwise Wilcoxon, FDR < 0.05).

Immune Scores and Overall Survival

Cell type	HR	Lower 95% CI	Upper 95% CI	p-value	C-index	FDR
Mast cells	0.885	0.831	0.942	<0.001	0.550	0.002
Macrophages	1.091	1.028	1.159	0.004	0.543	0.031
B cells	0.931	0.872	0.993	0.029	0.512	0.102
Treg	1.071	1.009	1.138	0.025	0.527	0.102
CD8 T cells	0.939	0.883	0.999	0.047	0.508	0.131
Cytotoxic cells	0.948	0.891	1.009	0.093	0.499	0.217
NK cells	1.051	0.988	1.119	0.115	0.510	0.230
Neutrophils	1.041	0.982	1.103	0.174	0.514	0.287
Th1 cells	1.042	0.980	1.108	0.185	0.515	0.287
CD4 T cells	1.025	0.968	1.086	0.395	0.512	0.554
Exhausted CD8	0.980	0.923	1.041	0.519	0.493	0.660
NK CD56dim	0.989	0.931	1.050	0.708	0.484	0.826
CD45	0.992	0.931	1.057	0.804	0.491	0.860
DC	1.006	0.945	1.070	0.860	0.518	0.860

NoteFigure 6 / Table 2 — Univariate Cox regression: immune scores and overall survival

Hazard ratios per SD increase from univariate Cox proportional hazards models for each immune cell type (FDR < 0.05).

Despite reaching FDR significance, the best-performing single cell type (Mast cells) achieved a concordance of only 0.55, indicating modest discriminative ability. Given the strong subtype dependence of immune infiltration observed in Q1, we next asked whether adjusting for PAM50 subtype alters the prognostic landscape.

PAM50-Adjusted Immune Prognostic Analysis

To account for subtype confounding, we fitted multivariable Cox models adjusting for PAM50 subtype for each of the 14 cell types. 4 cell types reached FDR < 0.05 after adjustment (B cells, Cytotoxic cells, CD8 T cells, Mast cells). For these, we additionally examined per-subtype effects.

Cell type	HR	Lower 95% CI	Upper 95% CI	p-value	FDR	C-index
B cells	0.889	0.830	0.952	<0.001	0.011	0.587
Cytotoxic cells	0.903	0.845	0.965	0.003	0.018	0.582
CD8 T cells	0.914	0.858	0.974	0.006	0.024	0.582
Mast cells	0.911	0.852	0.975	0.007	0.024	0.584
NK CD56dim	0.925	0.865	0.989	0.022	0.061	0.579
Treg	1.066	1.003	1.132	0.038	0.089	0.582
Exhausted CD8	0.942	0.883	1.005	0.069	0.139	0.578
NK cells	1.056	0.992	1.123	0.086	0.151	0.578
Neutrophils	1.046	0.987	1.108	0.132	0.205	0.581
Th1 cells	1.045	0.983	1.110	0.160	0.208	0.580
Macrophages	1.045	0.982	1.111	0.164	0.208	0.582
CD45	0.973	0.913	1.037	0.394	0.459	0.573
DC	0.978	0.917	1.043	0.499	0.537	0.575
CD4 T cells	1.006	0.949	1.066	0.852	0.852	0.577

NoteFigure 7 / Table 3 — PAM50-adjusted Cox regression and per-subtype effects

Cox proportional hazards models adjusted for PAM50 subtype. Forest plot shows the 4 cell types reaching FDR < 0.05; table shows all 14.

Interaction models (immune score × PAM50 subtype) revealed no significant heterogeneity for B cells (p = 0.047, FDR = 0.189), Cytotoxic cells (p = 0.250, FDR = 0.333), CD8 T cells (p = 0.443, FDR = 0.443), Mast cells (p = 0.102, FDR = 0.205), suggesting that while the magnitude of the prognostic effect varies by subtype, the direction is broadly consistent.

NoteFigure 8 — Kaplan-Meier survival curves for PAM50-adjusted significant cell types

Patients split at median score. P-values from log-rank test; titles show PAM50-adjusted HR and FDR.

Aggregating Immune Scores via Clustering

NoteFigure 9 — Immune cluster centroids

Mean z-scored immune cell type profiles for k-means clusters. Left: 2 clusters from all 14 cell types. Right: 2 clusters from the 4 PAM50-adjusted significant cell types only. Clusters labelled by overall immune level. Red = above-average infiltration; blue = below-average.

Model	df	Concordance	AIC	LR p vs PAM50	ΔAIC
PAM50 subtype only	4	0.576	14057.6	—	0.0
PAM50 + B cells	5	0.587	14047.5	<0.001	-10.1
PAM50 + Cytotoxic cells	5	0.582	14050.3	0.002	-7.3
PAM50 + CD8 T cells	5	0.582	14051.8	0.005	-5.8
PAM50 + Mast cells	5	0.584	14052.2	0.007	-5.4
PAM50 + Cluster (4 sig, k=2)	5	0.577	14056.6	0.086	-0.9
PAM50 + Cluster (all 14, k=2)	5	0.576	14058.5	0.314	1.0

NoteTable 4 — Prognostic comparison: PAM50 baseline vs immune-augmented models

LR test p-values against PAM50-only baseline. ΔAIC relative to baseline (negative = better fit).

Discussion

Conclusion

References

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Addendum

Even if my day job is somewhat cancer adjacent, diving deep into this METABRIC dataset made me feel like a novice when it came to cancer / breast cancer. But it has been fun seeing what we learn in class come together in more ways than one. The lead authors for the immune cell scoring paper are from the company NanoString Technologies, the same company that has the commercial PAM50 test kit that we learned in class. And in the same paper, they cite the false discovery rate (FDR) correction paper from Benjamini and Hochberg!

This whole assignment has been a rollercoaster ride, and there were so many rabbit holes that this topic would lead you into. For example Danaher didn’t make it easy for me when they tweaked the TIL acronym, usually meaning tumour infiltrating lymphocytes, to sneak in leukocytes in the palce of lymphocytes, so that the immune cells of myeloid origin could be included in the paper. The inclusion of Claudin-low